Field of the Invention
The present invention relates to the detection of a target nucleic acid sequence by a POCH (PO Cleavage and Hybridization) assay on a solid substrate.
Description of the Related Art
DNA hybridization-based technologies would be a very useful tool in specific nucleic acid sequence determination and clearly be valuable in clinical diagnosis, genetic research, and forensic laboratory analysis. Besides probe hybridization processes, several approaches using additional enzymatic reactions, for example, TaqMan™ probe method, have been suggested.
In TaqMan™ probe method, the labeled probe hybridized with target nucleic acid sequences is cleaved by a 5′ nuclease activity of an upstream primer-dependent DNA polymerase and a labeled fragment is released (U.S. Pat. Nos. 5,210,015, 5,538,848 and 6,326,145). The release of the labeled fragment indicates cleavage of the probe, finally indicating the presence of target sequences. The detection of the labeled fragment may be performed by size analysis such as gel electrophoresis, sedimentation in gradients, gel exclusion chromatography and homochromatography. The cleavage of probes may be carried out in a real-time manner by use of interactive dual labels.
The TaqMan™ probe method suggests two approaches for signal generation: polymerization-dependent cleavage and polymerization-independent cleavage. In polymerization-dependent cleavage, extension of the upstream primer must occur before a nucleic acid polymerase encounters the 5′-end of the labeled probe. As the extension reaction continues, the polymerase progressively cleaves the 5′-end of the labeled probe. In polymerization-independent cleavage, the upstream primer and the labeled probe are hybridized with a target nucleic acid in close proximity such that binding of the nucleic acid polymerase to the 3′-end of the upstream primer puts it in contact with the 5′-end of the labeled probe to release the label. In addition, the TaqMan™ probe method discloses that the labeled probe at its 5′-end having a 5′-tail region not-hybridizable with target sequences is also cleaved to form a fragment comprising the 5′-tail region.
There have been reported some methods in which probes having a 5′-tail region non-complementary to target sequences are cleaved by 5′ nuclease to release a fragment comprising the 5′-tail region and target detection is performed using the fragment comprising the 5′-tail region.
For instance, U.S. Pat. No. 5,691,142 discloses a cleavage structure to be digested by 5′ nuclease activity of DNA polymerase. The cleavage structure is exemplified in which an oligonucleotide comprising a 5′ portion non-complementary to and a 3′ portion complementary to a template is hybridized with the template and an upstream oligonucleotide is hybridized with the template in close proximity. The cleavage structure is cleaved by DNA polymerase having 5′ nuclease activity or modified DNA polymerase with reduced synthetic activity to release the 5′ portion non-complementary to the template. The released 5′ portion is then hybridized with an oligonucleotide having a hairpin structure to form a cleavage structure, thereby inducing progressive cleavage reactions to detect target sequences.
U.S. Pat. No. 7,381,532 discloses a process in which the cleavage structure having the upstream oligonucleotide with blocked 3′-end is cleaved by DNA polymerase having 5′ nuclease activity or FEN nuclease to release non-complementary 5′ flap region and the released 5′ flap region is detected by size analysis or interactive dual label.
U.S. Pat. Appln. Pub. 2008-0241838 discloses a target detection method using cleavage of single-labeled probes having non-complementary 5′ portion to target and capture probes immobilized on solid substrates. A single label is positioned on the non-complementary 5′ portion of the labeled probe. The labeled probes hybridized with target are cleaved to release fragments, after which the fragments are then hybridized with the capture probes to detect the presence of the target sequence. In this method, it is necessary that an uncleaved/intact probe is not hybridized with the capture probe. For this performance, the method prevents the uncleaved target probe from hybridizing with the immobilized capture-probe by controlling immobilization orientation of the immobilized oligonucleotide and its distance from the surface of a solid substrate. However, such limitation results in lower efficiency of hybridization on a solid substrate and difficulties in optimization of reaction conditions.
U.S. Pat. Appln. Pub. 2008-0193940 also discloses a target detection method using probes having non-complementary sequence (tag or flap sequence) to target as well as capture probes immobilized on solid substrates. A label is also positioned on the non-complementary region of probes. Undigested probes form hairpin structure and is not hybridized with capture probes. In contrast, where probes are digested, label-containing fragments are then hybridized with capture probes, thereby detecting the presence of target nucleic acid sequences. However, it has serious problems in which reaction conditions has to be elaborately controlled in considering Tm value of hybridization between target sequences and probes, and also considering Tm value of hairpin structure of undigested probes as well as Tm value of hybridization between digested fragments and capture probes.
Therefore, there remain long-felt needs in the art to develop novel approaches for detection of a target sequence on a solid phase, particularly, being free from shortcomings of conventional technologies using tag sequence-carrying probes and capturing probes immobilized onto solid substrates.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.